Connection fragment PCR detecting method for appraising unknown gene sequence

A technology for connecting fragments and detection methods, which is applied in the field of unknown DNA sequences, can solve the problems of obtaining coding sequences on functional genes, failing to obtain regulatory element sequences, consuming a lot of manpower and material resources, and achieving amplification specificity and amplification. Efficiency enhancement, simple and reliable result analysis, simplified labor effect

Inactive Publication Date: 2008-04-02
CHINA AGRI UNIV
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Problems solved by technology

Random sequencing is a large number of randomly determined DNA sequences, and the method of sorting with the help of computer power to obtain the required DNA sequence, there is a lot of chance, and it usually consumes a lot of manpower and material resources
There is another method called RACE, which can help to obtain the full-length mRNA sequence of the target gene under the condition that the partial mRNA sequence is known, but RACE cannot obtain the upstream and downstream regulatory element sequences of a gene, only Obtain the coding sequence on the functional gene
Since the premise of the success of RACE is that the reaction of mRNA reverse transcription into cDNA sequence should be well controlled, but because mRNA is easy to degrade, the reverse transcription reaction is also particularly vulnerable to low abundance and high-order structure.
The method of using isotope or biotin-labeled probes to hybridize and screen DNA libraries is even more time-consuming and laborious, and is the last choice for researchers when they have to

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  • Connection fragment PCR detecting method for appraising unknown gene sequence
  • Connection fragment PCR detecting method for appraising unknown gene sequence

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Embodiment 1

[0030] Amplification of the insertion site for transgenic crop GT73. In this experiment, the restriction endonuclease Hind III was selected to digest the rapeseed genome, and the digested DNA was ligated with a junction fragment with a Hind III restriction site sticky end under the action of ligase, and then the exogenous gene cp4- The nested forward primer of the epsps gene and the nested reverse primer on the connecting fragment were used for nested PCR amplification of the connected system. After two rounds of PCR amplification, a very bright electrophoresis band greater than 2000 bp was obtained. Collect the amplified product, connect the recovered fragment to the pGEM-T Easy vector, transform Escherichia coli competent cell DH5α, pick positive clones, digest and sequence to identify the obtained sequence. Some of the measured sequences are shown in Figure 3. According to DNAMAN and NCBI analysis, the sequence before 2372bp is derived from exogenously inserted DNA, and th...

Embodiment 2

[0032]Amplification of plant unknown sequences next to the insertion site in transgenic maize 59122. In this experiment, the restriction endonuclease Sac I was selected to digest the rapeseed genome, and the digested DNA was ligated with a junction fragment with a sticky end of the Sac I restriction site under the action of ligase, and then the exogenous gene promoter of transgenic crops was used The nested primers (UbiF1 / R1 / R2) of the gene and the nested primers (SS1 / SS2) on the connecting fragment were used to perform nested PCR amplification on the connected system. After two rounds of PCR amplification, a very bright Electrophoretic bands larger than 2000bp. The amplified product was recovered, and the recovered fragment was connected to the pGEM-T Easy vector, transformed into Escherichia coli competent cell DH5α, positive clones were picked, digested with restriction enzymes, and sequenced to identify the obtained sequence. . Align the known sequences with DNAMAN softw...

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Abstract

The present invention provides a method to identify the unknown sequences on both sides of a known DNA sequence, and carries out the restriction digestion on the sites of restriction endonucleases for the known sequence, collects the product of restriction digestion, which is ligated into a specific sequence (S sequence) with the sites of restriction endonucleases. The ligated product is amplified with two sets of siv specific primers, the positive one of which adopts respectively universal promoter sequences or terminator sequences from a sequenced segment, or a target gene situating near the unknown boundary, wherein, the negative one is selected from a segment of specific sequence with only one terminus situated with sites of specific restriction digestion. The method uses the primers as above to carry out PCR amplification, the product of which undergoes primary identification with siv primers to obtain the segments, which are finally cloned and then sequenced. The present invention transforms the amplification of the unknown sequence into the amplification of the known one through introducing a specific sequence, and has simple technical proposal, fewer limits on the operation, easy and reliable analytical result, and low cost.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for determining the unknown DNA sequence on both sides of the DNA or gene sequence from a part of the known DNA (Deoxyribonucleic Acid) sequence by using the connection fragment technology and the PCR (Polymerase Chain Reaction) technology. Background technique [0002] The characteristic of modern biology is that without knowing the complete DNA sequence of a gene, it is impossible to conduct in-depth research on the structure and function of the gene, and quite a few of the new genes that have been reported so far have only obtained partial DNA sequences. The problem often encountered in research work is to identify the complete DNA sequence of the gene from the known partial DNA sequence, especially the 5'upstream and 3'downstream DNA sequences including various regulatory elements. The current methods of identifying unknown sequences at both ends based on known ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许文涛黄昆仑罗云波白卫滨元延芳
Owner CHINA AGRI UNIV
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